Although all cells in the human body contain the same genetic material, the same genes are not active in all of those cells. Alterations in gene expression patterns can have profound effects on biological functions. These variations in gene expression are at the core of altered physiologic and pathologic processes. Therefore, identifying and quantifying the expression of genes in normal cells compared to diseased cells can aid the discovery of new drug and diagnostic targets.
Nucleic acids can be detected and quantified based on their specific polynucleotide sequences. The basic principle underlying existing methods of detection and quantification is the hybridization of a labeled complementary probe sequence to a target sequence of interest in a sample. The formation of a duplex indicates the presence of the target sequence in the sample.
This technique, called molecular hybridization, has been a useful tool for identifying and analyzing specific nucleic acid sequences in complex mixtures. This technique has been used in diagnostics, for example, to detect nucleic acid sequences of various microbes in biological samples. In addition, hybridization techniques have been used to map genetic differences or polymorphisms between individuals. Furthermore, these techniques have been used to monitor changes in gene expression in different populations of cells or in cells treated with different agents.
The identification of small RNA molecules, microRNA (miRNA) molecules, with hybridization techniques presents several unique challenges. Hybridization of a detection probe to a short length RNA molecule of a typical miRNA target occurs at low melting temperatures and prevents concurrent binding by multiple detection moeities. Low melting temperature hybridizations are unfavorable for specific binding of multiple probes to target miRNA sequences that may differ by only a single nucleic acid. Furthermore, miRNA sequences demonstrate great diversity despite having constant or conserved lengths, which generates a large variety of hybridization melting temperatures.
Thus, there exists a need for accurate and sensitive detection, identification and quantification of target nucleic acid molecules in complex mixtures. Particularly, there exists a need for the specific detection of small RNA molecules, such as miRNA molecules, in complex mixtures or multiplex reactions.